Title |
Directed analysis of cyanobacterial membrane phosphoproteome using stained phosphoproteins and titanium-enriched phosphopeptides§ |
Author |
Dong-Gi Lee1, Joseph Kwon1, Chi-Yong Eom2, Young-Moon Kang3, Seong Woon Roh1, Kyung-Bok Lee1, and Jong-Soon Choi1,3* |
Address |
1Biological Disaster Analysis Group, Korea Basic Science Institute, Daejeon 305-806, Republic of Korea, 2Molecular Imaging Group, Korea Basic Science Institute, Seoul 120-140, Republic of Korea, 3Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea |
Bibliography |
Journal of Microbiology, 53(4),279-287, 2015,
|
DOI |
10.1007/s12275-015-5021-8
|
Key Words |
cyanobacterial membrane phosphoprotein, phospho-tagging, phospho-trapping, FT-MS |
Abstract |
Gel-free shotgun phosphoproteomics of unicellular cyanobacterium
Synechocystis sp. PCC 6803 has not been reported
up to now. The purpose of this study is to develop directed
membrane phosphoproteomic method in Synechocystis sp.
Total Synechocystis membrane proteins were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and phosphoprotein-stained gel bands were selectively subjected
to in-gel trypsin digestion. The phosphorylation sites
of the resulting peptides were determined by assigning the
neutral loss of [M-H3PO4] to Ser, Thr, and Tyr residues using
nano-liquid chromatography 7 Tesla Fourier transform mass
spectrometry. As an initial application, 111 proteins and 33
phosphoproteins were identified containing 11 integral membrane
proteins. Identified four unknown phosphoproteins
with transmembrane helices were suggested to be involved in
membrane migration or transporters based on BLASTP search
annotations. The overall distribution of hydrophobic amino
acids in pTyr was lower in frequency than that of pSer or
pThr. Positively charged amino acids were abundantly revealed
in the surrounding amino acids centered on pTyr. A
directed shotgun membrane phosphoproteomic strategy provided
insight into understanding the fundamental regulatory
processes underlying Ser, Thr, and Tyr phosphorylation in
multi-layered membranous cyanobacteria. |